Keiichi Kanno

High night temperature stimulates photosynthesis, biomass production and growth during the vegetative stage of rice plants

Abstract The effects of night temperature on biomass accumulation and plant morphology were examined in rice (Oryza sativa L.) during vegetative growth. Plants were grown under three different night temperatures (17, 22 and 27°C) for 63 days. The day temperature was maintained at 27°C in all treatments. The final biomass of the plants was greatest in the plants grown at the highest night temperature. Total leaf area and tiller number were also the greatest in this treatment. Growth analysis indicated that the relative growth rate in the 27°C night-temperature treatment was maximal between days 21–42 and this was caused by increases in leaf area ratio, leaf weight ratio and specific leaf area. Plant total nitrogen contents did not differ among treatments. However, nitrogen allocation to the leaf blades was highest and the accumulation of sucrose and starch in the leaf blades and sheaths was the lowest in the 27°C night-temperature treatment by day 42. Despite this, dark respiration was also highest, and both the gross and net rates of CO2 uptake at the level of the whole plant at day 63 were the highest in the 27°C night-temperature treatment. Thus, high night temperature strongly stimulated the growth of leaf blades during the early stage of rice plant growth, leading to increased biomass during the vegetative stage of the rice plants. As the CO2 uptake rate per total leaf area was higher, photosynthesis at the level of the whole plant was also stimulated by a high night temperature.

Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice

Characterization of a rice mutant defective in autophagy highlights its importance in nitrogen remobilization from senescent leaves, biomass increase, and nitrogen use efficiency in the vegetative plant. Much of the nitrogen in leaves is distributed to chloroplasts, mainly in photosynthetic proteins. During leaf senescence, chloroplastic proteins, including Rubisco, are rapidly degraded, and the released nitrogen is remobilized and reused in newly developing tissues. Autophagy facilitates the degradation of intracellular components for nutrient recycling in all eukaryotes, and recent studies have revealed critical roles for autophagy in Rubisco degradation and nitrogen remobilization into seeds in Arabidopsis (Arabidopsis thaliana). Here, we examined the function of autophagy in vegetative growth and nitrogen usage in a cereal plant, rice (Oryza sativa). An autophagy-disrupted rice mutant, Osatg7-1, showed reduced biomass production and nitrogen use efficiency compared with the wild type. While Osatg7-1 showed early visible leaf senescence, the nitrogen concentration remained high in the senescent leaves. 15N pulse chase analysis revealed suppression of nitrogen remobilization during leaf senescence in Osatg7-1. Accordingly, the reduction of nitrogen available for newly developing tissues in Osatg7-1 likely led its reduced leaf area and tillers. The limited leaf growth in Osatg7-1 decreased the photosynthetic capacity of the plant. Much of the nitrogen remaining in senescent leaves of Osatg7-1 was in soluble proteins, and the Rubisco concentration in senescing leaves of Osatg7-1 was about 2.5 times higher than in the wild type. Transmission electron micrographs showed a cytosolic fraction rich with organelles in senescent leaves of Osatg7-1. Our results suggest that autophagy contributes to efficient nitrogen remobilization at the whole-plant level by facilitating protein degradation for nitrogen recycling in senescent leaves.

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content.

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.

Asparagine synthetase1, but not asparagine synthetase2, is responsible for the biosynthesis of asparagine following the supply of ammonium to rice roots.

Asparagine is synthesized from glutamine by the reaction of asparagine synthetase (AS) and is the major nitrogen form in both xylem and phloem sap in rice (Oryza sativa L.). There are two genes encoding AS, OsAS1 and OsAS2, in rice, but the functions of individual AS isoenzymes are largely unknown. Cell type- and NH4(+)-inducible expression of OsAS1 as well as analyses of knockout mutants were carried out in this study to characterize AS1. OsAS1 was mainly expressed in the roots, with in situ hybridization showing that the corresponding mRNA was specifically accumulated in the three cell layers of the root surface (epidermis, exodermis and sclerenchyma) in an NH4(+)-dependent manner. Conversely, OsAS2 mRNA was abundant in leaf blades and sheathes of rice. Although OsAS2 mRNA was detectable in the roots, its content decreased when NH4(+) was supplied. Retrotransposon-mediated knockout mutants lacking AS1 showed slight stimulation of shoot length and slight reduction in root length at the seedling stage. On the other hand, the mutation caused an approximately 80-90% reduction in free asparagine content in both roots and xylem sap. These results suggest that AS1 is responsible for the synthesis of asparagine in rice roots following the supply of NH4(+). Characteristics of the NH4(+)-dependent increase and the root surface cell-specific expression of OsAS1 gene are very similar to our previous results on cytosolic glutamine synthetase1;2 and NADH-glutamate synthase1 in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4(+) in rice roots.

Effect of individual suppression of RBCS multigene family on Rubisco contents in rice leaves

In higher plants, a small subunit of Rubisco is encoded for by an RBCS multigene family in the nuclear genome. However, it is unknown how each multigene member contributes to the accumulation of Rubisco holoenzyme. Here, four RBCS genes that are highly expressed in leaf blaedes of rice (Oryza sativa L.) were individually suppressed by RNAi, and the effects on leaf Rubisco content were examined at seedling, vegetative and reproductive stages. Rubisco contents in each transgenic line declined irrespective of growth stage, and the ratios of Rubisco-N to total N were 66-96% of wild-type levels. The mRNA levels of the suppressed RBCS genes declined significantly, whereas those of the unsuppressed ones did not change drastically. These results indicate that four RBCS genes all contribute to accumulation of Rubisco holoenzyme irrespective of growth stage and that suppression of one RBCS gene is not fully compensated by other RBCS genes. Additionally, the mRNA levels of the large subunit of Rubisco showed a change similar to that of total RBCS mRNA level irrespective of genotype and growth stage. These results suggest that gene expression of RBCS and RBCL is regulated in a coordinated manner at the transcript level in rice.

Lack of cytosolic glutamine synthetase1;2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings.

The development and elongation of active tillers in rice was severely reduced by a lack of cytosolic glutamine synthetase1;2 (GS1;2), and, to a lesser extent, lack of NADH-glutamate synthase1 in knockout mutants. In situ hybridization using the basal part of wild-type seedlings clearly showed that expression of OsGS1;2 was detected in the phloem companion cells of the nodal vascular anastomoses and large vascular bundles of axillary buds. Accumulation of lignin, visualized using phloroglucin HCl, was also observed in these tissues. The lack of GS1;2 resulted in reduced accumulation of lignin. Re-introduction into the mutants of OsGS1;2 cDNA under the control of its own promoter successfully restored the outgrowth of tillers and lignin deposition to wild-type levels. Transcriptomic analysis using a 5 mm basal region of rice shoots showed that the GS1;2 mutants accumulated reduced amounts of mRNAs for carbon and nitrogen metabolism, including C1 unit transfer in lignin synthesis. Although a high content of strigolactone in rice roots is known to reduce active tiller number, the reduction of outgrowth of axillary buds observed in the GS1;2 mutants was independent of the level of strigolactone. Thus metabolic disorder caused by the lack of GS1;2 resulted in a severe reduction in the outgrowth of axillary buds and lignin deposition.

Increased grain yield and biomass allocation in rice under cool night temperature

Abstract The effects of different night temperatures on grain yield were examined in rice (Oryza sativa L. Akita-63) during the ripening period. Plants were grown under two different night temperatures (22 and 27°C) from anthesis to harvesting. The day temperature was maintained at 27°C in both treatments. Although the final biomass at harvest did not differ between the treatments, the dry weight of the panicles was significantly greater in the cool night temperature treatment. This increase in panicle weight was associated with increases in the 1000-kernel weight and the ratio of filled spikelets. Although panicle respiration in the high night temperature treatment decreased to almost zero just after the cessation of grain filling, the net CO2 fixation rate per day in the whole plant in this treatment tended to be higher, and this was associated with a higher level of starch accumulation. After grain filling, the starch content decreased and the final dry weight of other plant parts, including dead organs and new tillers, was greater in the high night temperature treatment. Thus, the noted decline in yield at the high night temperature led to changes in carbon allocation to new sinks for vegetative reproduction at the whole plant level. From these results, we considered the possibility that a cool night temperature tends to favor carbon allocation to panicles, resulting in higher yield.

Contributions of two cytosolic glutamine synthetase isozymes to ammonium assimilation in Arabidopsis roots

Highlight Temporal and spatial distributions of GLN1;2 and GLN1;3, the two low-affinity cytosolic glutamine synthetase isoforms, determine their contributions to ammonium assimilation in Arabidopsis roots.

Production of 1-m size uniform plasma by modified magnetron-typed RF discharge with a subsidiary electrode for resonance

Abstract A large-diameter uniform plasma of 1 m in size is produced using a modified magnetron-typed (MMT) RF plasma source at the frequency of 13.56 MHz. The construction and operation of the MMT RF plasma source are very simple and we can place two substrates simultaneously. To achieve an efficient production of high density plasma, a parallel resonance circuit is connected to one of the substrates which acts as a subsidiary RF electrode controlling the plasma parameters. In the case of the resonance the plasma density increases to approximately three times as much as that in case of non-resonance. The plasma density reaches∼1×10 11 /cm 3 in Ar at 1 mtorr when the RF input power is 2.8 kW. The MMT RF plasma source provides a plasma with uniformity within several percent over 1 m in diameter in front of the substrate in the low gas pressure regime.

Growth Properties and Biomass Production in the Hybrid C4 Crop Sorghum bicolor

Hybrid vigor (heterosis) has been used as a breeding technique for crop improvement to achieve enhanced biomass production, but the physiological mechanisms underlying heterosis remain poorly understood. In this study, to find a clue to the enhancement of biomass production by heterosis, we systemically evaluated the effect of heterosis on the growth rate and photosynthetic efficiency in sorghum hybrid [Sorghum bicolor (L.) Moench cv. Tentaka] and its parental lines (restorer line and maintainer line). The final biomass of Tentaka was 10-14 times greater than that of the parental lines grown in an experimental field, but the relative growth rate during the vegetative growth stage did not differ. Tentaka exhibited a relatively enlarged leaf area with lower leaf nitrogen content per leaf area (Narea). When the plants were grown hydroponically at different N levels, daily CO2 assimilation per leaf area (A) increased with Narea, and the ratio of A to Narea (N-use efficiency) was higher in the plants grown at low N levels but not different between Tentaka and the parental lines. The relationships between the CO2 assimilation rate, the amounts of photosynthetic enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase, Chl and Narea did not differ between Tentaka and the parental lines. Thus, Tentaka tended to exhibit enlargement of leaf area with lower N content, leading to a higher N-use efficiency for CO2 assimilation, but the photosynthetic properties did not differ. The greater biomass in Tentaka was mainly due to the prolonged vegetative growth period.